Compositions and methods for upconverting luminescence with engineered excitation and applications thereof

ABSTRACT

The invention generally relates to materials and methods for creating and/or utilizing upconverting luminescence. More particularly, the invention relates to novel compositions (e.g., nanoparticles) and related methods of preparation and use that enable upconverting luminescence with an efficient excitation optimized at about 800 nm. A unique class of cascade sensitized tri-doped UCNPs with a biocompatiable 800 nm excitable property are disclosed herein, for example, tri-doped β-NaYF4:Nd, Yb, Er (Tm)/NaYF4UCNPs, which employ Nd3+ as 800 nm photon sensitizer and Yb3+ as bridging ions, having strong green or blue upconversion emissions without photobleaching.

PRIORITY CLAIMS AND RELATED PATENT APPLICATIONS

This application is a divisional application of and claims the benefitof priority to U.S. Ser. No. 14/761,546, filed Jul. 16, 2015, which isthe U.S. national phase of PCT/US2014/012437, filed Jan. 22, 2014, whichclaims the benefit of priority to U.S. Provisional Application No.61/755,424, filed Jan. 22, 2013, the entire content of each of which isincorporated herein by reference in its entirety.

TECHNICAL FIELDS OF THE INVENTION

The invention generally relates to materials and methods for generatingand/or utilizing upconverting luminescence. More particularly, theinvention relates to novel materials and compositions (e.g.,nanoparticles) and related methods of preparation and use that enableupconverting luminescence with an efficient and optimized excitation(e.g., at about 800 nm).

BACKGROUND OF THE INVENTION

Upconversion nanoparticles (UCNPs) have recently emerged as a new classof materials with potential applications in a wide-range of fields, suchas biosensing, chemical sensing, in vivo imaging, drug delivery,photodynamic therapy and photoactivation. (Zhan, et al. 2011 Acs Nano 5,3744; Wang, et al. 2005 Angew Chem Int Edit 44, 6054; Achatz, et al.2011 Angew Chem Int Edit 50, 260; Liu, et al. 2011 Acs Nano 5, 8040;Liu, et al. 2011 J Am Chem Soc 133, 17122; Chen, et al. 2012 Acs Nano 6,8280; Lim, et al. 2006 Nano Lett 6, 169; Wang, et al. 2011 Biomaterials32, 1110; Hou, et al. 2011 Adv Funct Mater 21, 2356; Tian, et al. 2012Adv Mater 24, 1226; Shan, et al. 2011 Adv Funct Mater 21, 2488; Zhang,et al 2007 J Am Chem Soc 129, 4526; Jayakumar, et al. 2012 Natl Acad SciUSA 109, 8483; Yang, et al. 2012 Angew Chem Int Edit 51, 3125; Yan, etal. 2012 J Am Chem Soc 134, 16558; U.S. Pat. Nos. 7,332,344; 7,790,392;7,501,092; 8,088,631.)

Upconverting luminescence refers to an anti-Stokes type process in whichthe sequential absorption of two or more photons leads to the emissionof light at shorter wavelength (e.g., ultraviolet, visible, andnear-infrared) than the excitation wavelength. For instance, Lanthanideion (Ln³⁺) doped UCNPs are able to absorb near-infrared (NIR) photonsand convert such low energy excitation into shorter wavelengthemissions. (Haase, et al. 2011 Angew Chem Int Edit 50, 5808.) Utilizinglong-lived, ladder-like energy levels of Ln³⁺, the intensity ofanti-Stokes luminescence of UCNPs is orders of magnitude more potentcompared with those of conventional synthetic dyes or quantum dots(QDs). (Wang, et al. 2009 Chem Soc Rev 38, 976; U.S. Provisional Appl.No. 61/675,019 by Han, et al.; U.S. Provisional Appl. No. 61/653,406 byHan, et al.; PCT/US13/42555 by Han, et al. filed May 24, 2013.)

Challenges remain, however, that hamper the wide use of UCNPs. Forexample, a major limitation of the most commonly used Yb³⁺-sensitizedUCNPs is their physically unalterable excitation band centered at 980 nm(the peak absorption of Yb³⁺ ions), overlapping with the maximumabsorption peak of water molecules (FIG. 1). Because cells and tissueswithhold 980 nm radiation and concomitantly induce heat damages, thisbecomes problematic for application of UCNPs in water-rich biologicalsystems. (McNichols, et al. 2004 Laser Slug Med 34, 48; Nam, et al. 2011Angew Chem Int Edit 50, 6093.) In particular, the heating effect islikely more severe where greater power density and longertermirradiation are required, such as in single nanoparticle imaging orlongitudinally deep tissue imaging.

While extensive research has resulted in continued progress in themodulation of UCNP's emissions, for example, via composing properdopants/matrix or FRET process, few studies have focused on engineeringthe excitation of UCNPs. (Tian, et al. 2012 Adv Mater 24, 1226; Wang, etal. 2011 Nat Mater 10, 968; Li, et al. 2008 Adv Mater 20, 4765; Yi, etal. 2011 Chem Mater 23, 2729; Chan, et al. 2012 Nano Lett 12, 3839;Heer, et al. 2003 Angew Chem Int Edit 42, 3179; Zhan, et al. 2011 AcsNano 5, 3744.) A recent report showed the use of an alternativeexcitation peak at 915 nm in Yb³⁺-sensitized cubic phase (α) NaYF₄:LnUCNPs. This excitation peak, however, is still well within the regime ofintrinsic absorption of Yb³⁺ dopants, which partially overlaps with theabsorption peak of water. Another report used dye-sensitized UCNPs under800 nm excitation, in which an antenna dye was used to stimulate theYb³⁺—Er³⁺ upconverting process via the fluorescence resonance energytransfer (FRET) mechanism. (Zou, et al. 2012 Nat Photonics 6, 560.) ThisFRET-based approach, however, is limited to the organic media with thesynthetic dyes susceptible to photo-bleaching. Additionally, the FRETprocess is restricted by the distance between the organic molecules andthe UCNPs.

Thus, un-met needs continue to exist for novel compositions and methodsthat enable upconverting luminescence with efficient excitation awayfrom peak absorption of water, preferably near a minimum of waterabsorption.

SUMMARY OF THE INVENTION

The invention provides novel upconverting luminescence materials (e.g.,UCNPs) and methods that have constitutional excitation engineered tooptimize at about 800 nm, which is not only well away from peakabsorption of water, but also is ideally situated at a local minimum ofwater absorption. A unique class of cascade sensitized tri-doped UCNPswith a biocompatiable 800 nm excitable property are disclosed herein,for example, tri-doped β-NaYF₄:Nd, Yb, Er (Tm)/NaYF₄ UCNPs, which employNd³⁺ as 800 nm photon sensitizer and Yb³⁺ as bridging ions, affordingstrong green or blue upconversion emissions without photobleaching. TheUCNPs of the invention are preferably configured, for example, in aβ-NaYF₄:Yb, Er (Tm)/NaYF₄, Yb, Nd core/shell or a β-NaYF₄:Yb, Er(Tm)/NaYF₄, Yb, Nd/NaYF₄ core/shell/shell architects. These UCNPs areamendable to physically engineering the excitation wavelengths ofupconversion nanoparticles and can be employed in a wide range ofapplications, for example, in biological sensing, chemical sensing, invitro or in vivo imaging (e.g., tumor-targeted imaging, multimodalimaging), drug delivery, photodynamic therapy, photoactivation,photovoltaic and solar cells, photocatalysts, and 3-D displays.

In one aspect, the invention generally relates to an upconversionluminescence material. The upconversion luminescence material includes:(1) a first Lanthanide, Ln_((i)), having a mol % from about 0.1% toabout 10% (e.g., 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%,6%, 7%, 8%, 9%); (2) a second Lanthanide ion, Ln_((j)), having a mol %from about 10% to about 80% (e.g., 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65% 70%, 75%); and (3) a third Lanthanide ion, Ln_((k)),having a mol % from about 0.1 to about 10% (e.g., 0.2%, 0.5%, 1%, 1.5%,2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, 9%). The upconversionluminescence material is characterized by a constitutional excitationpeak substantially away from 980 nm. In certain preferred embodiments,the upconversion luminescence material is characterized by aconstitutional excitation peak at about 800 nm, the upconversionluminescence material includes β-NaYF₄, and Ln_((i)) is Nd; Ln_((j)) isYb; and Ln_((k)) is Er or Tm.

In another aspect, the invention generally relates to a biocompatibleupconversion luminescence nanoparticle, which include: a tri-doped coreof β-NaYF₄: Ln_((i)), Ln_((j)), Ln_((k)); and an epitaxial shell ofβ-NaYF₄, wherein Ln_((i)) is Nd; Ln_((j)) is Yb; and Ln_((k)) is Er, Hoor Tm.

In yet another aspect, the invention generally relates to abiocompatible upconversion luminescence nanoparticle, which includes: acore of β-NaYF₄:Ln_((j)), Ln_((k)); an epitaxial inner shell ofβ-NaYF₄:Ln_((i)), Ln_((j)), Ln_((k)); and an epitaxial outer shell ofβ-NaYF₄. Ln_((i)) is Nd; Ln_((j)) is Yb; and Ln_((k)) is Er or Tm.

In yet another aspect, the invention generally relates to a sensingprobe for detecting a target molecule in a sample. The sensing probeincludes an upconversion luminescence material and/or a biocompatibleupconversion luminescence nanoparticle disclosed herein.

In yet another aspect, the invention generally relates to a method fordetecting a target molecule in a sample. The method includes: providinga sensing probe comprising an upconversion luminescence nanoparticlehaving a constitutional excitation peak at about 800 nm, wherein thesensing probe is capable of association with the target molecule;contacting the sensing probe with a sample to be tested for the presenceof the target molecule under a condition such that if the targetmolecule is present the sensing probe becomes associated with the targetmolecule; exciting the sensing probe with an excitation at about 800 nm;and detecting an emission at a shorter wavelength than 800 nm todetermining the presence of the target molecule.

In yet another aspect, the invention generally relates to an imagingsystem comprising an upconversion luminescence material and/or anupconversion luminescence nanoparticle of the invention.

In yet another aspect, the invention generally relates to a photonicssystem comprising an upconversion luminescence material and/or aupconversion luminescence nanoparticle of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Spectra profiles showing tissue optical window. The extinctioncoefficient of water at 800 nm is about 20 timers lower than that at 980nm.

FIG. 2. Upconversion process of Nd³⁺→Yb³⁺→Er³⁺(Tm³⁺) tri-dopants systemwith 800 nm excitation.

FIG. 3. Transmission Electron Microscopy (TEM) images of (a)β-NaYF₄:0.5% Nd, 20% Yb, 2% Er, (b) β-NaYF₄:Nd, Yb, Er/NaYF₄, (c)β-NaYF₄:1% Nd, 30% Yb, 0.5% Tm and (d) β-NaYF₄:Nd, Yb, Tm/NaYF₄UCNPs.(e) The X-ray Diffraction (XRD) patterns of four samples and index dataof β-NaYF₄.

FIG. 4. The upconverting emission spectra and emission counts summary of(a, b) β-NaYF₄:(0-5%) Nd, 20% Yb, 2% Er/NaYF₄ and (c, d) β-NaYF₄:(0-3%)Nd, 30% Yb, 0.5% Tm/NaYF₄ UCNPs. The measurement was applied under 800nm CW laser excitation (6.0 W/cm²) using the concentration normalizedUCNPs solutions. The upconverting luminescent pictures were inserted in(b, d) with the laser path labeled.

FIG. 5. The upconverting emission spectra and emission counts of (a, b,c) β-NaYF₄:Nd, Yb, Er core and core/shell UCNPs, (d, e, f) β-NaYF₄:Nd,Yb, Tm core and core/shell UCNPs under 800 nm CW laser excitation (6.0W/cm²). All of the UCNPs solutions have the same particleconcentrations.

FIG. 6. (a) Hydrodynamics size and (b) ζ potential distributions of PAAmodified β-NaYF₄:0.5% Nd, 20% Yb, 2% Er/NaYF₄ and β-NaYF₄:1% Nd, 30% Yb,0.5% Tm/NaYF₄ UCNPs.

FIG. 7. The output power of (a) the 800 nm CW laser and (b) the 980 nmlaser under different operating laser currents.

FIG. 8. β-NaYF₄:0.5% Nd, 20% Yb, 2% Er (left) and the correspondingcore/shell (right) UCNPs. Their respective statistical sizes are20.2±1.6 nm and 29.5±1.0 nm.

FIG. 9. β-NaYF₄:1% Nd, 20% Yb, 2% Er (left) and the correspondingcore/shell (right) UCNPs. Their respective statistical sizes are20.4±0.6 nm and 29.2±0.9 nm.

FIG. 10. β-NaYF₄:2% Nd, 20% Yb, 2% Er (left) and the correspondingcore/shell (right) UCNPs. Their statistical sizes are 20.3±1.2 nm and29.0±1.3 nm.

FIG. 11. β-NaYF₄:3% Nd, 20% Yb, 2% Er (left) and the correspondingcore/shell (right) UCNPs. Their respective statistical sizes are19.7±0.7 nm and 29.3±1.2 nm.

FIG. 12. β-NaYF₄:5% Nd, 20% Yb, 2% Er (left) and the correspondingcore/shell (right) UCNPs. Their respective statistical sizes are19.9±1.4 nm and 28.8±0.9 nm.

FIG. 13. β-NaYF₄:1% Nd, 40% Yb, 2% Er (left) and the correspondingcore/shell (right) UCNPs. Their respective statistical sizes are21.2±1.0 nm and 29.4±1.4 nm.

FIG. 14. β-NaYF₄:1% Nd, 60% Yb, 2% Er (left) and the correspondingcore/shell (right) UCNPs. Their respective statistical sizes are20.5±0.8 nm and 29.1±2.3 nm.

FIG. 15. β-NaYF₄:20% Yb, 2% Er (left) and the corresponding core/shell(right) UCNPs. Their respective statistical sizes are 20.7±0.7 nm and29.8±0.5 nm.

FIG. 16. β-NaYF₄:0.5% Nd, 30% Yb, 0.5% Tm (left) and the correspondingcore/shell (right) UCNPs. Their respective statistical sizes are20.0±1.1 nm and 29.1±0.5 nm.

FIG. 17. β-NaYF₄:1% Nd, 30% Yb, 0.5% Tm (left) and the correspondingcore/shell (right) UCNPs. Their respective statistical sizes are20.5±1.9 nm and 29.1±1.2 nm.

FIG. 18. β-NaYF₄:2% Nd, 30% Yb, 0.5% Tm (left) and the correspondingcore/shell (right) UCNPs. Their respective statistical sizes are20.1±1.7 nm and 29.3±1.8 nm.

FIG. 19. β-NaYF₄:1% Nd, 60% Yb, 0.5% Tm (left) and the correspondingcore/shell (right) UCNPs. Their respective statistical sizes are20.4±1.3 nm and 29.8±1.9 nm.

FIG. 20. β-NaYF₄:30% Yb, 0.5% Tm (left) and the corresponding core/shell(right) UCNPs. Their respective statistical sizes are 20.5±0.8 nm and29.2±0.9 nm.

FIG. 21. β-NaYF₄:2% Er (left) and the corresponding core/shell (right)UCNPs. Their respective statistical sizes are 20.4±0.7 nm and 29.3±0.7nm.

FIG. 22. β-NaYF₄:1% Nd (left) and the corresponding core/shell (right)UCNPs. Their respective statistical sizes are 20.7±0.5 nm and 29.9±0.8nm.

FIG. 23. β-NaYF₄:0.5% Nd, 2% Er (left) and the corresponding core/shell(right) UCNPs. Their respective statistical sizes are 20.4±0.6 nm and29.6±1.1 nm.

FIG. 24. (a) The Stokes emission spectra of β-NaYF₄:1% Nd/NaYF₄NPs under800 nm excitation. (b) The upconversion emission spectra of β-NaYF₄:20%Yb, 2% Er/NaYF₄, β-NaYF₄:2% Er/NaYF₄ and β-NaYF₄:1% Nd, 2% Er/NaYF₄UCNPs. This figure indicates that Yb³⁺ had little contribution to UCNPsphoton absorbency at 800 nm, and Nd³⁺ had deactivation effect to Er³⁺even at a low doping ratio. The power density of the 800 nm CW laser is6.0 W/cm². All of the UCNPs' solutions had similar particleconcentrations (0.57 μmol/L).

FIG. 25. The upconversion emission spectra of (a) core β-NaYF₄:(0-5%)Nd, 20% Yb, 2% Er, (b) core/shell β-NaYF₄:(0-5%) Nd, 20% Yb, 2%Er/NaYF₄, (d) core β-NaYF₄:(0-3%) Nd, 30% Yb, 0.5% Tm, and (e)core/shell β-NaYF₄:(0-3%) Nd, 30% Yb, 0.5% Tm/NaYF₄ UCNPs under 800 nmCW laser. Their corresponding emission counts are summarized in (c) and(f). The excitation power density is 6.0 W/cm². All of the UCNPssolutions had the same particle concentrations (0.57 μmol/L).

FIG. 26. (a, b) Upconversion power dependence of β-NaYF₄:0.5% Nd, 20%Yb, 2% Er/NaYF₄ and (c, d) β-NaYF₄:1% Nd, 30% Yb, 0.5% Tm/NaYF₄ (below)UCNPs under 800 nm CW laser excitation.

FIG. 27. The upconversion emission spectra of (a) core β-NaYF₄:(0-5%)Nd, 20% Yb, 2% Er, (b) core/shell β-NaYF₄:(0-5%) Nd, 20% Yb, 2%Er/NaYF₄, (d) core β-NaYF₄:(0-3%) Nd, 30% Yb, 0.5% Tm, and (e)core/shell β-NaYF₄:(0-3%) Nd, 30% Yb, 0.5% Tm/NaYF₄ UCNPs under 980 nmCW laser. Their corresponding emission counts were summarized in (c) and(f). The excitation power density is 23.5 W/cm². All UCNPs solutions hadthe same particle concentrations (0.57 μmol/L).

FIG. 28. Upconversion power dependence of (a) β-NaYF₄:(0%, 0.5%) Nd, 20%Yb, 2% Er/NaYF₄ and (b) β-NaYF₄:(0%, 1%) Nd, 30% Yb, 0.5% Tm/NaYF₄ UCNPsunder 980 nm CW laser excitation.

FIG. 29. The photo-stability test of β-NaYF₄:0.5% Nd, 20% Yb, 2%Er/NaYF₄ and β-NaYF₄:1% Nd, 30% Yb, 0.5% Tm/NaYF₄ UCNPs in hexanesolution under 800 nm CW laser excitation (6.0 W/cm²). The monitoringemission peaks are (a) ⁴S_(3/2)→⁴I_(15/2) (540 nm) of Er³⁺ and (b)¹G₄→³H₆ (474 nm) of Tm³⁺.

FIG. 30. Upconversion luminescence yield comparison under the same powerdensity 980 nm and 800 nm laser excitation (6 W/cm²). (a) Comparing 800nm excited β-NaYF₄:0.5% Nd, 20% Yb, 2% Er/NaYF₄ with 980 nm excitedβ-NaYF₄:20% Yb, 2% Er/NaYF₄ UCNPs. (b) Comparing 800 nm excitedβ-NaYF₄:1% Nd, 30% Yb, 0.5% Tm/NaYF₄ with 980 nm excited β-NaYF₄:30% Yb,0.5% Tm/NaYF₄ UCNPs. All of the UCNPs' solutions had the same particleconcentrations (0.57 μmol/L).

FIG. 31. Photographs of visible luminescence signals from core/shellUCNPs and CdSe/ZnS QDs solutions. The solutions of QDs, β-NaYF₄:(0%,0.5%) Nd, 20% Yb, 2% Er/NaYF₄ UCNPs under (a) 6 W/cm² of 800 nm CW laserand (b) 23.5 W/cm² of 980 nm CW laser excitation. The solutions of QDs,β-NaYF₄:(0%, 1%) Nd, 30% Yb, 0.5% Tm/NaYF₄ UCNPs under (c) 6 W/cm² of800 nm CW laser and (d) 23.5 W/cm² of 980 nm CW laser excitation. (e)The solutions of CdSe/ZnS core/shell QDs under 362 nm UV lampexcitation.

FIG. 32 TEM images of a β-NaYF₄:Yb, Er (Tm)/NaYF₄, Yb, Nd core/shell ora β-NaYF₄: Yb, Er (Tm)/NaYF₄, Yb, Nd/NaYF₄ core/shell/shell.

FIG. 33 The emission spectra of β-NaYF₄:Yb, Er (Tm)/NaYF₄, Yb, Ndcore/shell or a β-NaYF₄:Yb, Er (Tm)/NaYF₄, Yb, Nd/NaYF₄ core-shell-shellUCNPs under excitation of an 800 nm CW diode laser.

DESCRIPTION OF THE INVENTION

This invention provides a unique class of cascade sensitized tri-dopedUCNPs with a biocompatiable 800 nm excitable property, corresponding toa local minimum of water absorption. Tri-doped β-NaYF₄:Nd, Yb, Er(Tm)/NaYF₄UCNPs, for example, employ Nd³⁺ as 800 nm photon sensitizerand Yb³⁺ as bridging ions, resulting in strong green or blueupconversion emissions.

FIG. 1 shows spectra tissue optical window. Commonly usedYb³⁺-sensitized UCNPs have their peak absorption of Yb³⁺ ions(excitation) centered at 980 nm, which overlaps with the maximumabsorption peak of water molecules. In contrast, the extinctioncoefficient of water at 800 nm, the local minima of water absorption, isabout 20 timers lower than that at 980 nm. Therefore, 800 nm has beenconsidered to be the ideal excitation wavelength with the least impacton biological tissues. (Kobayashi, et al. 2010 Chem Rev 110, 2620; Xie,et al. 2012 Nat Mater 11, 842.)

The present invention discloses an unconventional strategy. Rather thanemploying dye-sensitization, the invention provides upconversionmaterials and UCNPs with constitutional excitation specificallyengineered to about 800 nm. This approach paves the way for broadapplications of a new generation of UCNPs with much-improvedbiocompatibility and exciting penetrability.

Unlike the single-type sensitizer (e.g., Yb³⁺) in typical dual dopedUCNPs, tri-doped cascade sensitized UCNPs of the invention employ aprimary sensitizer (e.g., Nd³⁺) and a secondary sensitizer (e.g., Yb³⁺).The probable upconversion mechanism with Nd³⁺ sensitizing and Yb³⁺transferring is presented in FIG. 2. Nd³⁺ ions take the role ofabsorbing photons at 800 nm, while the Yb³⁺ ions act as bridging ionsfor the energy transfer from the Nd³⁺ ions to the emitters, Er³⁺ orTm³⁺.

UCNPs prepared according to the methods of the invention, for example,Nd³⁺/Yb³⁺/Er³⁺(Tm³⁺) tri-doped core/shell β-NaYF₄ UCNPs prepared via thesolution synthesis, displayed robust upconverting emission with 800 nmcontinuous-wave (CW) laser excitation. The presence of a small quantityof Nd³⁺ (doping ratio≤1%) in the tri-doped systems is important for thepurpose of sensitizing at 800 nm, resulting in more than 20-foldstronger emissions than the traditional dual-dopants Yb³⁺-sensitizedsystem.

Significantly, with surface modification, the tri-doped UCNPs dispersedin water still exhibit strong 800 nm excitable luminescence, which isvisible to the naked eye in ambient indoor light. Along with theirnon-bleaching advantage, such tri-doped UCNPs are a new and promisingclass of anti-Stokes luminescent probes using biocompatible 800 nmexcitations.

More specifically, Nd³⁺ is known for its optical activity in the MRregion, and its ⁴I_(9/2)→⁴F_(5/2) transition offers a strong absorptionat ca. 800 nm. (Guyot, et al. 1995 Phys Rev B Condens Matter 51, 784.)It has been reported that the excited Nd³⁺ on the ⁴F_(5/2) level canrelax to the lower ⁴F_(3/2) level and then sensitize the ground stateYb³⁺ ions nearby via resonance energy transfer. (Balda, et al. 2010 OptExpress 18, 13842.) For example, previously, in glass ceramics and bulkcrystal materials, spectroscopic physics studies have experimentallyvalidated the feasiblity of such a 800 nm pumped upconversion process.(Lu, et al. 2007 J Lumin 126, 677; Chen, et al. 2007 Opt Lett 32, 3068;Li, et al. 2009 J Appl Phys 105, 013536.) However, unlike colloidaldispersible nanoparticles, due to their size dimensions, uncontrolledmorphology, low upconversion efficiency, and surface chemistry, thesebulk materials are rather problematic for biological usages. (Auzel 2004Chem Rev 104, 139; Lin, et al. 2010 J Appl Phys 107; Camargo, et al 2004J Appl Phys 95, 2135.) Yet, it has been a great challenge to achieveNd³⁺ sensitized strong upconversion inside small colloidalmonodispersible UCNPs. The possible reasons for this are as follows: (1)Compared with the Yb³⁺/Er³⁺ or Yb³⁺/Tm³⁺ dual-dopants combination, thecascade multiple-step resonance energy transfer (i.e., Nd³⁺→Yb³⁺→Er³⁺ orTm³⁺) of the tri-dopant upconversion systems suffer from a greatersurface quenching risk in the colloidal UCNPs. (2) Compared to thesimple excited state of Yb³⁺, the excited Nd³⁺ states are rathercomplicated for a primary sensitizer, and this may lead to more severedeleterious deactivation of the excited emitters via cross-relaxations.

The invention addresses the first challenge by novel configurations ofthe UCNPs, for example by way of epitaxial core/shell orcore/shell/shell strategies. Due to the advantage of low phonon energy,a hexagonal phased (β)-NaYF₄ matrix was selected. (Haase, et al. 2011Angew Chem Int Edit 50, 5808; Wang, et al. 2009 Chem Soc Rev 38, 976.) Amajor deleterious factor in regard to luminescence emission of colloidalUCNPs is the energy traps on their surface, which include sublatticedefects and external deactivators (e.g., ligands). The calculation for a20 nm β-NaYF₄:Ln nanosphere revealed that 29% of the total Ln³⁺ dopantsare exposed on the particle surface and are susceptible to such energydeactivation. Compared to Yb³⁺/Er³⁺ and Yb³⁺/Tm³⁺ dual-dopant systems,the tri-dopant's cascade sensitized upconversion undergoes additionalenergy transfer steps, i.e. the initial steps of Nd³⁺→Yb³⁺ sensitizing.If each Ln³⁺ has the same intralattice deactivation probability, theoverall yield of Nd³⁺→Yb³⁺→Er³⁺(Tm³⁺) upconversion will be exponentiallylower than that of Yb³⁺→Er³⁺(Tm³⁺). Thus, shielding energy deactivatorsfrom UCNP surface dopants is essential in the cascade sensitizedupconversion.

The typically adopted strategy of UCNPs surface passivation is that ofdeveloping an inert shell free of dopants. (Qian, et al. 2008 Langmuir24, 12123; Yi, et al. 2007 Chem Mater 19, 341.) Under the protection ofan epitaxial growth β-NaYF₄ shell, the UCNPs surface related quenchingcan be largely suppressed, and the efficiency differences between ourtri-dopants UCNPs and the classical dual-dopant UCNPs are shortened.(Su, et al. 2012 J Am Chem Soc 134, 20849.)

The solution-phase synthesis of β-NaYF₄:Nd, Yb, Er (Tm)/NaYF₄ core/shellUCNPs employed a modified trifluoroacetates thermolysis method. (Mai, etal. 2006 J Am Chem Soc 128, 6426.) It is worthy of noting that it is thefirst time that β-NaYF₄:Ln/NaYF₄ core/shell UCNPs were successfullyprepared with the trifluoroacetates thermolysis method. In brief,α-NaYF₄:Ln was first prepared as the intermediate UCNPs bythermo-decomposition of trifluoroacetate precursors in high-boilingpoint solvents at 300° C. This was followed by α→β phase-transition withadditional sodium trifluoroacetate at a higher crystallizationtemperature (325° C.). After purification, the as-prepared β-NaYF₄:LnUCNPs were reacted with fresh CF₃COONa and Y(CF₃COO)₃ for epitaxialgrowth of β-NaYF₄ shell via seed-mediated crystallization.

It has been reported that epitaxial shell thickness shows a directproportion to the emission enhancing folds on core/shell UCNPs.(Johnson, et al. 2012 J Am Chem Soc 134, 11068.) Since only a sufficientepitaxial growth can produce high-crystallized shell to protect UCNPcore in a compact manner, we set 1:2 ratio as the precursor's inputratio for the UCNP core and shell components. As shown in the TEM imagesfrom FIG. 3 and FIGS. 8-23, the β-NaYF₄:Nd, Yb, Er (Tm) core UCNPs havea uniform nanosphere morphology with an average diameter of 20 nm, andthe core/shell UCNPs have an average diameter of 29 nm. The calculatedvolume ratio of core and shell is ca. 1:2.05, well consistent with theinput ratio. The XRD patterns of core and core/shell UCNPs confirm thepresence of a pure p phase structure, while the core/shell UCNPs showslightly narrower peak bandwidths.

The invention addresses the second challenge by optimizing the primarysensitizer Nd³⁺ doping ratio in the tri-dopant upconversion system.Distinct from single excited state of Yb³⁺, Nd³⁺ has abundant energylevels that may deactivate emitter dopants rather than sensitize them.In order to weaken such deleterious deactivation, Nd³⁺ should be kept ata sufficient distance away from Er³⁺(Tm³⁺). Since all Ln³⁺ dopants areconsidered to be homogenously dispersed inside β-NaYF₄ matrix, theirmean distances have an inverse proportion to their concentrations. Thus,for optimization, Nd³⁺ was adjusted to a relatively low dopingconcentration (0.5-5%) inside UCNP core. (Chen, et al. 2012 Acs Nano 6,2969.) At the same time, for the other two dopant concentrations insidethe UCNPs core, the classical dual-dopants optimal ratios were followed,i.e. 20%/2% for Yb³⁺/Er³⁺, and 30%/0.5% for Yb³⁺/Tm³⁺. (Haase, et al.2011 Angew Chem Int Edit 50, 5808; Wang, et al. 2009 Chem Soc Rev 38,976.)

The upconvesion spectra were characterized on a SPEX Fluoromax-3spectrofluorimeter equipped with an 800 nm CW laser. All of theas-synthesized core and core/shell UCNPs were dispersed in hexane toform transparent colloidal solutions with the same particleconcentrations. Since Yb³⁺ ions have no intrinsic absorbency at ca. 800nm, β-NaYF₄:20% Yb, 2% Er/NaYF₄ and β-NaYF₄:30% Yb, 0.5% Er/NaYF₄ dualdoped core/shell UCNPs were employed as reference samples in themeasurement, and are denoted as 0% Nd³⁺ doping. The emission spectra andintensity counts of β-core/shell UCNPs are displayed in FIG. 4.Significantly, 0.5% of Nd³⁺ doped UCNPs was found to be the preferredconcentration and showed a 28.7-fold enhancement on the green emissionpeak of the Er³⁺ as compared with the none-Nd³⁺ samples. Such emissiongradually decreased as the Nd³⁺ concentration rose to 5%. This indicatesthat the increase of doped Nd³⁺ will quench the upconverting process,even when the total photon absorbance at 800 nm is raised.

To further confirm the essential roles of the Nd³⁺ and Yb³⁺ in thecascade sensitization upconversion pathway, two other control sampleswere synthesized: single doped β-NaYF₄:2% Er/NaYF₄ and non-Yb³⁺ dualdoped β-NaYF₄:1% Nd, 2% Er/NaYF₄ UCNPs. (FIG. 24). Without Nd³⁺sensitizing, it was found that upconversion intensity of β-NaYF₄:2%Er/NaYF₄ was as weak as that of β-NaYF₄:20% Yb,2% Er/NaYF₄ UCNPs under800 nm excitation. This demonstrates the 800 nm-insensibility of Yb³⁺ions and the essentiality of Nd³⁺ as the initiator ions in the cascadepathway.

In contrast, without Yb³⁺ bridging, Nd³⁺ only showed deleteriousquenching effects to Er³⁺ rather than useful sensitizing, even thoughtheir energy levels overlaps. Thus, the three types of dopants areintegral parts of the cascade sensitization system. The Nd³⁺/Yb³⁺/Tm³⁺upconversion system was also optimized, with the best enhancement of23.7-fold from β-NaYF₄:1% Nd, 30% Yb, 0.5% Tm/NaYF₄ core/shell UCNPs.This is likely due to a lower activator ratio (Tm³⁺ 0.5%) used in theNd³⁺/Yb³⁺/Tm³⁺ system, and the optimal Nd³⁺ ratio increased slightly to1%. FIGS. 4b and 4d show the optimized β-NaYF₄:Nd, Yb, Er (Tm)/NaYF₄core/shell UCNP solutions. Under a 6.0 W/cm² of 800 nm CW laserexcitation, the green (Er³⁺, ²H_(11/2), ⁴S_(b 3/2)→⁴I_(15/2)) and blue(Tm³⁺, ¹G₄→³H₆) upconverting emissions were clearly observed.

A solution of CdSe/ZnS QDs was placed at the side of the UCNPs solutionsas the control. Although QDs are known as excellent multi-photon probesvia pulsed laser excitation, there are no detectable emissions from QDsunder such a low power 800 nm CW laser. Moreover, it was observed thatβ-NaYF₄ shell coating was able to provide more than a 20-foldenhancement of β-NaYF₄:Nd, Yb, Er and a 50-fold enhancement ofβ-NaYF₄:Nd, Yb, Tm core UCNPs (FIG. 25). Thus, such a core/shellstructure is important in ensuring the success of multiple-stepresonance energy transfer in tri-doped colloid UCNPs. (Su, et al. 2012 JAm Chem Soc 134, 20849.)

The fitted slope of 800 nm excitation power dependence was 1.79 for the⁴S_(3/2)→⁴I_(15/2) transitions of Er³⁺ (2-photon upconversion) and 2.27for the ¹G₄→³H₆ transition of Tm³⁺ (3-photon upconversion) (FIG. 26).The rigorous linearity denoted that no excitation saturation occurred.As shown in FIG. 28, the slope values were similar to those of a typicaldual-dopant upconversion process under a 980 nm CW laser.

In regard to the secondary sensitizer, the Yb³⁺ ratios were investigatedin order to optimize the efficiencies of bridging energy transfer. Asshown in FIG. 5, increasing of Yb³⁺ ratio showed no improvement in theEr³⁺(Tm³⁺) emission yields. The result suggests that 20-30% of Yb³⁺dopants are sufficient to deliver energy from 1% doped Nd³⁺. On theother hand, it was found that the β-NaYF₄:Nd, Yb, Er (Tm)/NaYF₄core/shell UCNPs still maintained their 980 nm excitable upconvertingluminescence, which relied only on the sensitizer of Yb³⁺ (FIG. 27).Taking the optimal β-NaYF₄:Nd, Yb, Er/NaYF₄ UCNPs as examples, the 980nm motivated emission intensity from 0.5% Nd³⁺ doped UCNPs was ca. 67.8%of the outputs from β-NaYF₄:Yb, Er/NaYF₄UCNPs (FIG. 27C), which can beclearly seen in ambient indoor light (FIG. 31).

To test photo-stability, the β-NaYF₄:Nd, Yb, Er (Tm)/NaYF₄ UCNPssolutions was continuously excited with an 800 nm CW laser for 1 hour.No photo-bleaching was observed (FIG. 29). For purposes of watersolubility and future bio-applications, the as-synthesized β-NaYF₄:Nd,Yb, Er (Tm)/NaYF₄ UCNPs with oleic acid capping were treated by ligandexchange. (Dong, et al. 2011 J Am Chem Soc 133, 998.) Modified UCNPswith Poly(acrylic acid) (PAA) coating can disperse well in water to formtransparent solutions, with an average hydrodynamic size of 36.7 nm and41.3 nm for β-NaYF₄:0.5% Nd, 20% Yb, 2% Er/NaYF₄ and β-NaYF₄:1% Nd, 30%Yb, 0.5% Tm/NaYF₄ UCNPs, respectively. As shown in FIG. 6, the 800 nmexcited upconverting luminescence from their water solutions can beeasily seen by the naked eye in room light.

Thus, in one aspect, the invention generally relates to an upconversionluminescence material. The upconversion luminescence material includes:(1) a first Lanthanide, Ln_((i)), having a mol % from about 0.1% toabout 5% (e.g., 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%); (2)a second Lanthanide ion, Ln_((j)), having a mol % from about 10% toabout 80% (e.g., 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%70%, 75%); and (3) a third Lanthanide ion, Ln_((k)), having a mol % fromabout 0.1 to about 10% (e.g., 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%). The upconversion luminescence material is characterized by aconstitutional excitation peak substantially away from 980 nm.

Ln here refers to the lanthanide (or lanthanoid), the fifteen metallicchemical elements with atomic numbers 57 through 71, from lanthanumthrough lutetium.

Lanthanide La Ce Pr Nd Pm Sm Eu Gd Tb Dy Ho Er Tm Yb Lu 57 58 59 60 6162 63 64 65 66 67 68 69 70 71

In certain preferred embodiments, the upconversion luminescence materialis characterized by a constitutional excitation peak at about 800 nm. Incertain preferred embodiments, the upconversion luminescence material isbiocompatible.

In certain preferred embodiments, Ln_((i)) is Nd; Ln_((j)) is Yb; andLn_((k)) is Er, Ho or Tm (preferably Er or Tm).

In certain preferred embodiments, the upconversion luminescence materialincludes β-NaYF₄, CaF₂, LiYF₄, NaGdF₄, NaScF₄, α-NaYF₄, NaYbF₄, NaLaF₄,LaF₃, GdF₃, GdOF, La₂O₃, Lu₂O₃, Y₂O₃, Y₂O₂S, YbF₃, YF₃, KYF₄, KGdF₄,BaYF₅, BaGdF₅, NaLuF₄, KLuF₄, BaLuF₅ or a mixture of two or more thereof(e.g., in the core and/or shell of nanoparticles).

In certain preferred embodiments, Nd, having a mol % from about 0.2 toabout 3% (e.g., from about 0.5% to about 2%); Yb, having a mol % fromabout 20% to about 70% (e.g., from about 20% to about 50%); and Er orTm, having a mol % from about 0.2 to about 5% (e.g., from about 0.2% toabut 3%).

In certain preferred embodiments, the upconversion luminescence materialis characterized by a nano-structure (e.g., in the form ofnanoparticles).

The nanoparticles may have any suitable dimensions, for example, with adimension the range from about 2 nm to about 150 nm (e.g., from about 2nm to about 100 nm, from about 3 nm to about 50 nm, from about 5 nm toabout 30 nm).

In certain preferred embodiments, the nanoparticles have a core/shellconfiguration, preferably an epitaxial configuration.

In certain preferred embodiments, the core includes β-NaYF₄:Nd, Yb, Eror β-NaYF₄:Nd, Yb, Tm; and the shell includes β-NaYF₄. In certainembodiments, the core has a dimension from about 1 nm to about 100 nm(e.g., about 2 nm, about 5 nm, about 10 nm, about 20 nm, about 30 nm,about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about90 nm) and the shell has a dimension from about 1 nm to about 50 nm(e.g., about 2 nm, about 5 nm, about 10 nm, about 20 nm, about 30 nm,about 40 nm). In certain preferred embodiments, the upconversionluminescence material is characterized by an emission spectrum in thevisible region, e.g., a peak in the green/yellow region (e.g., at about540 nm, at about 525 nm) or a peak in the blue region (e.g., at about474 nm).

In certain preferred embodiments, the nanoparticles have acore/shell/shell configuration comprising an inner shell and an outershell, preferably in an epitaxial configuration.

In certain preferred embodiments, the core includes β-NaYF₄:Yb,Tm; theinner shell includes β-NaYF₄:Nd, Yb, Er or β-NaYF₄:Nd, Yb, Tm; and theshell include β-NaYE₄.

In certain preferred embodiments, the core has a dimension from about 5nm to about 100 nm (e.g., from about 5 nm to about 80 nm, from about 5nm to about 60 nm, from about 5 nm to about 50 nm, from about 5 nm toabout 30 nm, from about 5 nm to about 20 nm, from about 10 nm to about100 nm, from about 20 nm to about 100 nm, from about 30 nm to about 100nm, from about 50 nm to about 100 nm), the inner shell has a dimensionfrom about 1 nm to about 20 nm (e.g., about 1 nm to about 15 nm, about 1nm to about 10 nm, about 1 nm to about 5 nm, about 2 nm to about 20 nm,about 5 nm to about 20 nm, about 10 nm to about 20 nm), and the outershell has a dimension from about 1 nm to about 20 nm (e.g., about 1 nmto about 15 nm, about 1 nm to about 10 nm, about 1 nm to about 5 nm,about 2 nm to about 20 nm, about 5 nm to about 20 nm, about 10 nm toabout 20 nm). In certain preferred embodiments, the upconversionluminescence material is characterized by an emission spectrumcomprising a peak at about 540 nm. In certain preferred embodiments, theupconversion luminescence material is characterized by an emissionspectrum in the visible region, e.g., a peak in the green/yellow region(e.g., at about 540 nm, at about 525 nm) or a peak in the blue region(e.g., at about 474 nm).

In another aspect, the invention generally relates to a biocompatibleupconversion luminescence nanoparticle, which include: a tri-doped coreof β-NaYF₄: Ln_((i)), Ln_((j)), Ln_((k))); and an epitaxial shell ofβ-NaYF₄, wherein Ln_((i)) is Nd; Ln_((j)) is Yb; and Ln_((k)) is Er, Hoor Tm.

The term “biocompatible”, as used herein, refers to a material that iscompatible with living cells, tissues, organs or systems, and posesminimal or no risk of injury, toxicity, or rejection by the immunesystem. A biocompatible material may be a synthetic or natural materialused to replace part of a living system or to function in intimatecontact with living tissue. Biocompatible materials typically interfacewith biological systems to evaluate, treat, augment or replace anytissue, organ or function of the body.

In certain preferred embodiments, the upconversion luminescence ischaracterized by a constitutional excitation peak at about 800 nm.

In certain embodiments, Nd accounts for a mol % from about 0.1% to about5% of the core (e.g., 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%,4.5%); Yb accounts for a mol % from about 10% to about 80% of the core(e.g., 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% 70%, 75%);and Er or Tm accounts for a mol % from about 0.1 to about 10% of thecore (e.g., 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%).

In certain preferred embodiments, Nd accounts for a mol % from about 0.2to about 3% of the core; Yb accounts for a mol % from about 20% to about70% of the core; and Er or Tm accounts for a mol % from about 0.2 toabout 5% of the core.

In certain preferred embodiments, the biocompatible upconversionluminescence nanoparticle is characterized by an emission spectrum inthe visible region, e.g., a peak at about 540 nm, at about 525 nm,and/or at about 474 nm).

The biocompatible upconversion luminescence nanoparticle may include achemically modified and/or fundtionalized surface, such as bypoly(acrylic acid), citric acid, and molecules with —COOH or —NH₂groups, for example. A variety of groups and chemistries are availablefor such functionalization. (Zhou, et al. 2012 Chem. Soc. Rev. 41,1323-1349.)

In yet another aspect, the invention generally relates to abiocompatible upconversion luminescence nanoparticle, which includes: acore of β-NaYF₄: Ln_((j)); an epitaxial inner shell of β-NaYF₄:Ln_((i)), Ln_((j)), Ln_((k)); and an epitaxial outer shell of β-NaYF₄.Ln_((i)) is Nd; Ln_((j)) is Yb; and Ln_((k)) is Er or Tm.

In certain preferred embodiments, the upconversion luminescence ischaracterized by a constitutional excitation peak at about 800 nm.

In certain embodiments, Nd accounts for a mol % from about 0.1% to about5% of the inner shell (e.g., 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%,4%, 4.5%); Yb accounts for a mol % from about 10% to about 80% of theinner shell (e.g., 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%70%, 75%); and Er or Tm accounts for a mol % from about 0.1 to about 10%of the inner shell (e.g., 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%).

In certain preferred embodiments, the core has a dimension from about 5nm to about 100 nm (e.g., from about 5 nm to about 80 nm, from about 5nm to about 60 nm, from about 5 nm to about 50 nm, from about 5 nm toabout 30 nm, from about 5 nm to about 20 nm, from about 10 nm to about100 nm, from about 20 nm to about 100 nm, from about 30 nm to about 100nm, from about 50 nm to about 100 nm), the inner shell has a dimensionfrom about 1 nm to about 20 nm (e.g., about 1 nm to about 15 nm, about 1nm to about 10 nm, about 1 nm to about 5 nm, about 2 nm to about 20 nm,about 5 nm to about 20 nm, about 10 nm to about 20 nm), and the outershell has a dimension from about 1 nm to about 20 nm (e.g., about 1 nmto about 15 nm, about 1 nm to about 10 nm, about 1 nm to about 5 nm,about 2 nm to about 20 nm, about 5 nm to about 20 nm, about 10 nm toabout 20 nm). In certain preferred embodiments, the upconversionluminescence material is characterized by an emission spectrumcomprising a peak at about 540 nm. In certain preferred embodiments, theupconversion luminescence material is characterized by an emissionspectrum in the visible region, e.g., a peak in the green/yellow region(e.g., at about 540 nm, at about 525 nm) or a peak in the blue region(e.g., at about 474 nm).

In yet another aspect, the invention generally relates to a sensingprobe for detecting a target molecule in a sample. The sensing probeincludes an upconversion luminescence material and/or a biocompatibleupconversion luminescence nanoparticle disclosed herein.

In yet another aspect, the invention generally relates to a method fordetecting a target molecule in a sample. The method includes: providinga sensing probe comprising an upconversion luminescence nanoparticlehaving a constitutional excitation peak at about 800 nm, wherein thesensing probe is capable of association with the target molecule;contacting the sensing probe with a sample to be tested for the presenceof the target molecule under a condition such that if the targetmolecule is present the sensing probe becomes associated with the targetmolecule; exciting the sensing probe with an excitation at about 800 nm;and detecting an emission at a shorter wavelength than 800 nm todetermining the presence of the target molecule.

The target molecule may be any suitable molecules. Exemplary targetmolecules include proteins (including peptides, antibodies, enzymes) andnucleic acids (including oligonucleotides). The biocompatibleupconversion luminescence nanoparticle can be chemically coupled to orotherwise associated with various target molecules.

In yet another aspect, the invention generally relates to an imagingsystem comprising an upconversion luminescence material and/or anupconversion luminescence nanoparticle of the invention.

In yet another aspect, the invention generally relates to a photonicssystem comprising an upconversion luminescence material and/or anupconversion luminescence nanoparticle of the invention. As used herein,the term “photonics” refers to the generation, emission, transmission,modulation, signal processing, switching, amplification, anddetection/sensing of light.

The invention disclosed herein allowed for the first time theconstitutional excitation wavelength of colloidal dispersible UCNPs tobe engineered. In particular, Nd³⁺/Yb³⁺/Er³⁺(Tm³⁺) tri-doped core/shellβ-NaYF₄ colloidal UCNPs with a constitutional biocompatiable 800 nmexcitable property were successfully developed. The cascade sensitizedtri-dopants upconversion nanoparticle system was well optimized forideal intra-Ln³⁺ energy transfer. The optimized β-NaYF₄:Nd, Yb, Er(Tm)/NaYF₄ UCNPs are characterized by a monodispersed size of about 29nm, and a more than 20-fold enhancement of emission yield under 800 nmCW laser excitation with no photo-bleaching. Significantly, upon surfacemodification, such upconversion luminescence in aqueous phase is stillfound to be easily visible to the naked eye in ambient light, showingcomparable efficiency with traditional 980 nm CW laser excitation. Thetri-dopants core/shell UCNPs of the invention thus open the door toengineering the excitation wavelengths of upconversion nanoparticles andprovide a powerful tool for a wide variety of applications in the fieldsof biophotoics and photonics.

EXAMPLES

Synthesis of β-NaYF₄:Ln Core UCNPs

The β-NaYF₄:Ln Core UCNPs were prepared by a two-step thermolysis method^([23]). In the first step, CF₃COONa (0.5 mmol) and proper Ln(CF₃COO)₃(0.5 mmol in total) precursors were mixed with oleic acid (5 mmol),oleyl amine (5 mmol) and 1-octadecene (10 mmol) in a two-neck reactionflask. The slurry mixture was heated to 110° C. to form a transparentsolution followed by 10 minutes of degassing. Then the flask was heatedto 300° C. with a rate of 15° C./min under dry argon flow, and itmaintained at 300° C. for 30 minutes. The α-NaYF₄:Ln intermediate UCNPswere gathered from the cooled reaction solution by centrifugal washingwith excessive ethanol. In the second step, the α-NaYF₄:Ln intermediateUCNPs were re-dispersed into oleic acid (10 mmol) and 1-octadecene (10mmol) together with CF₃COONa (0.5 mmol) in a new two-neck flask. Afterdegassing at 110° C. for 10 minutes, this flask was heated to 325° C.with a rate of 15° C./min under dry argon flow, and remained at 325° C.for 30 minutes. Then, β-NaYF₄:Ln UCNPs were centrifugally separated fromthe cooled reaction media and preserved in hexane (10 mL) as stocksolution.

Synthesis of β-NaYF₄:Ln/NaYF₄ Core/Shell UCNPs

The as-synthesized β-NaYF₄:Ln UCNPs served as core nanoparticles forepitaxial growth of the undoped β-NaYF₄ shell. Typically, 5.0 mL of theβ-NaYF₄:Ln UCNPs stock solution (ca. 0.25 mmol of total Ln³⁺) wastransferred into a two-neck flask and the hexane was evaporated byheating. Then CF₃COONa (0.5 mmol) and Y(CF₃COO)₃ (0.5 mmol) wereintroduced as β-NaYF₄ shell precursors together with solvents of oleicacid (10 mmol) and 1-octadecene (10 mmol). After 10 minutes of degassingat 110° C., the flask was heated to 325° C. at a rate of 15° C./minunder argon protection, and was maintained at 325° C. for 30 minutes.The products can be precipitated by adding ethanol to the cooledreaction flask. After centrifugal washing with hexane/ethanol, thecore/shell UCNPs were re-dispersed in hexane (10 mL).

Surface Modification for Water-Soluble Core/Shell UCNPs

The hydrophobic oleic acid coated β-NaYF₄:Ln/NaYF₄UCNPs were transferredinto water using a modified NOBF₄ treating route. ( ) Dong, et al. 2011J Am Chem Soc 133, 998. In the first step, nitrosonium tetrafluoroborate(NOBF₄, 0.20 g) was dissolved in dimethylformamide (DMF, 5 mL), and theβ-NaYF₄:Ln/NaYF₄UCNPs in hexane stock solution (1 mL) were added,followed by 2 h of stirring in a sealed pot at room temperature. Thenthe BF₄ ⁻ capped UCNPs were precipitated by adding an isopropanol/hexanemixture at 1:1 volume ratio, and purified by 2 cycles of centrifugalwash with DMF. In the second step, all of the UCNPs precipitate weredispersed in Poly(acrylic acid) (PAA, M_(w) 1800) solution (PAA 100 mg,DMF 10 mL) to replace surface BF₄ ⁻ with PAA. After overnightincubation, the PAA coated UCNPs were purified by centrifugal wash withDI water, and dispersed in 5 mL of DI water.

Synthesis of Core/Shell/Shell 800 nm Excitable UCNPs

In this UCNP architect, for example, β-NaYF₄:(20-99.5%) Yb, 0.5%Tm/β-NaYF₄(20-80%) Nd, 10% Yb/β-NaYF₄ core/shell/shell UCNPs, Nd³⁺concentration can be elevated, thus providing improved upconversionemission outcomes.

Typical synthetic protocols are described below. Exemplary TEM images ofcore-shell and core-shell-shell are shown in FIG. 32. The emissionspectra of core-shell and core-shell-shell UCNPs under excitation of an800 nm CW diode laser are shown in FIG. 33.

1. Synthesis Protocol for β-NaYF₄:99.5% Yb, 0.5% Tm Core UCNPs

The β-NaYF₄:99.5% Yb, 0.5% Tm Core UCNPs were prepared using a modifiedtwo-step thermolysis method. (Mai, et al. 2006 J. Am. Chem. Soc. 128,6426.) In the first step, the CF₃COONa (1 mmol) and required Ln(CF₃COO)₃(0.5 mmol in total, 99.5% Yb, 0.5% Tm) precursors were mixed with oleicacid (5 mmol), oleyl amine (5 mmol) and 1-octadecene (10 mmol) in atwo-neck reaction flask. The slurry mixture was heated to 110° C. inorder to form a transparent solution. This was followed by 10 minutes ofdegassing to remove the oxygen and water. The flask was then heated to300° C. at a rate of 15° C./min under dry argon flow, and remained at300° C. for 30 minutes. The α-NaLnF₄ intermediate UCNPs were acquired bycooling down the reaction solution to room temperature, followed bycentrifugation with excessive ethanol. In the second step, the α-NaYF₄:99.5% Yb, 0.5% Tm UCNPs were re-dispersed in oleic acid (10 mmol) and1-octadecene (10 mmol) along with CF₃COONa (0.5 mmol) in a two-neckflask. After degassing at 110° C. for 10 minutes, the flask was heatedto 325° C. at a rate of 15° C./min under dry argon flow, and remained at325° C. for 30 minutes. The β-NaYF₄:Yb,Tm UCNPs were then centrifugallyseparated from the cooled reaction media and suspended in 10 mL ofhexane as the stock solution for further use.

2. Synthesis of β-NaYF₄:99.5% Yb, 0.5% Tm@NaYF₄:50% Nd, 10% Yb(Core/Shell) UCNPs

In this thermolysis reaction, as-synthesized β-NaYF₄: 99.5% Yb, 0.5% TmUCNPs served as crystallization seeds for the epitaxial growth ofundoped β-NaYF₄ shell. Typically, a stock solution of β-NaYF₄:Yb, TmUCNPs (5 mL, ca. 0.26 μmol/L core UCNPs) was transferred into a two-neckflask and hexane was sequentially removed by heating. Then CF₃COONa (0.5mmol) and Ln(CF₃COO)₃ (0.5 mmol) were introduced as UCNP shellprecursors with oleic acid (10 mmol) and 1-octadecene (10 mmol). After10 minutes of degassing at 110° C., the flask was heated to 325° C. at arate of 15° C./min under dry argon flow and was kept at 325° C. for 30minutes. The products were precipitated by adding ethanol to the cooledreaction flask. After centrifugal washing with hexane/ethanol, thecore/shell UCNPs were re-dispersed in 10 mL of hexane for spectracharacterization.

3. Synthesis of β-NaYF₄:99.5% Yb, 0.5% Tm@NaYF₄:50% Nd, 10%Yb@NaYF₄(Core/Shell/Shell) UCNPs

The synthetic procedure of β-NaYF₄:99.5% Yb,0.5% Tm@NaYF₄:50% Nd, 10%Yb@NaYF₄ was the same as that used to synthesize β-NaYF₄: 99.5% Yb, 0.5%Tm@NaYF₄:50% Nd, 10% Yb UCNPs nanocrystals, except that 0.5 mmolCF₃COONa, 0.5 mmol Y(CF₃COO)₃ and the 10 ml β-NaYF₄:99.5% Yb, 0.5%Tm@NaYF₄:50% Nd nanocrystals prepared were added to a mixture of OA (10mmol) and ODE (10 mmol) in a three-necked flask. The finalcore-shell-shell UCNPs were re-dispersed in 10 mL of hexane for spectracharacterization.

Materials

Y₂O₃ (99.9%), Nd₂O₃ (99.9%), Yb₂O₃ (99.9%), Er₂O₃ (99.9%), Tm₂O₃(99.9%), CF₃COONa (99.9%), CF₃COOH, 1-octadecene (90%), oleic acid(90%), oleyl amine (90%), were all purchased from Sigma-Aldrich and usedwithout further purification. The lanthanide (Ln) trifluoroacetates,Ln(CF₃COO)₃, were prepared as literature described. (Roberts 1961 J AmChem Soc 83, 1087) CdSe/ZnS quantum dots (QE=45%, λ_(em) 530 nm) weresynthesized as described previously. (Talapin, et al. 2001 Nano Lett 1,207.)

The Volume Ratio of Core and Shell Components

The average diameters of sphere-like core and core/shell UCNPs are 20 nm(d) and 29 nm (D) respectively, so the approximative volume ratio ofinner β-NaYF₄:Nd, Yb, Er (Tm) core and outer β-NaYF₄ shell can beexpressed by the following formula:V _(core) /V _(shell) =πd ³/6÷(πD ³/6−πd ³/6)=d ³/(D ³ /d ³)=1:2.05UCNPs Concentration

In this research, all core and core/shell UCNP samples had monodispersesize distributions. The epitaxial growth of undoped β-NaYF₄ shell wassupposed to perform relatively fixed output yields for all core UCNPs,together with the uniform diameter of core (ca. 20 nm) and core/shell(ca. 29 nm) UCNPs, and the consistent value between the precursors inputratio and core/shell volume ratio. Thus, it can be concluded that alltypes of β-core/shell UCNP samples have the similar synthesis yields andtheir final stock solutions had the similar particle concentrations.

The approximate volume of a β-NaYF₄ unit cell (Na_(1.5)Y_(1.5)F₆,a=0.596 nm, c=0.353 nm) single core/shell particle (D=29 nm) can becalculated with the following formulas:V ₀=3^(1/2) ×a ² ×c÷2=0.1086 nm³V _(UCNP) =πD ³/6=12763 nm³Therefore, the approximate amount of Ln³⁺ ions in a single β-core/shellnanosphere can be calculated with the following formulas:Ln³⁺=1.5×V _(UNCP) /V ₀=1.18×10⁵If we assume the synthesis yield is 90%, the approximate particleconcentrations in β-core/shell UCNPs stock solutions can be calculatedwith the following formulas:c=(0.25+0.5) mmol×90%/118000÷10 mL=0.57 μmol/LSurface Ln³⁺ Proportion on β-NaLnF₄ UCNPs

The amount of Ln³⁺ ions in the outermost layer of unit cells of UCNPs isestimated using the following approach. A hexagonal phased (β) NaYF₄unit cell prism has the following parameters: a=0.596 nm, c=0.353 nm.The length of its longer body diagonal can be calculated with thefollowing formula:d=(c ²+3×a ²)^(1/2)=1.091 nm.

Due to the global curvature of the UCNPs, it is complicated to count allof the unit cells that are exposed on the UCNP surface. A simpler way isto define the thickness of a continual layer of these outmost unit cellsand then calculate the sum volume. Considering the disorder and defectsin the lattice packing on the UCNP surface, we defined the length of das the reasonable thickness of the outmost unit cells layer. Therefore,the proportion of surface Ln³⁺ can be calculated by the followingequation:n _(surface) /n _(tatol) =V _(surface) /V _(tatol)=[r ³−(r−d)³]/r ³where r is the radius of the spherical UCNPs. For a 20 nm β-NaLnF₄UCNP,we can estimate that its surface Ln³⁺ account is ca. 29% in total Ln³⁺quantity. Since all dopants are homogeneously dispersed in β-NaLnF₄nanosphere, the surface expose probability is also 29% for each kind ofLn³⁺ dopant.Spectra Characterization

All upconversion luminescence spectra were measured by a SPEXFluoromax-3 spectrofluorimeter (Horiba) (spectral resolution of 0.5 nm,emission slit of 1 nm, and integral period of 0.2 s) that was equippedwith a continuous wave (CW) laser. The MR laser was introduced into thespectrofluorimeter chamber using an optical fiber and focused by aconvex lens. The approximate diameter of the focused laser beam is 1.8mm, fitted by a pinhole (beam cross-section area 0.025 cm²). The totallaser power at the cuvette site was measured by a Newport co. opticalpower meter. For the sake of quantifying emission intensities, all UCNPsin hexane solution had the same particle concentrations (0.57 μmol/L).For power dependence study, the laser power was modulated by currentcontrol in the measurement.

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference, unless the context clearlydictates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although any methods and materials similar or equivalent tothose described herein can also be used in the practice or testing ofthe present disclosure, the preferred methods and materials are nowdescribed. Methods recited herein may be carried out in any order thatis logically possible, in addition to a particular order disclosed.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made in this disclosure. All such documents arehereby incorporated herein by reference in their entirety for allpurposes. Any material, or portion thereof, that is said to beincorporated by reference herein, but which conflicts with existingdefinitions, statements, or other disclosure material explicitly setforth herein is only incorporated to the extent that no conflict arisesbetween that incorporated material and the present disclosure material.In the event of a conflict, the conflict is to be resolved in favor ofthe present disclosure as the preferred disclosure.

EQUIVALENTS

The representative examples are intended to help illustrate theinvention, and are not intended to, nor should they be construed to,limit the scope of the invention. Indeed, various modifications of theinvention and many further embodiments thereof, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the full contents of this document, including the examples andthe references to the scientific and patent literature included herein.The examples contain important additional information, exemplificationand guidance that can be adapted to the practice of this invention inits various embodiments and equivalents thereof.

What is claimed is:
 1. A method for detecting a target molecule in asample, comprising: providing a sensing probe comprising an upconversionluminescence nanoparticle having a constitutional excitation peak atabout 800 nm, wherein the sensing probe is capable of association withthe target molecule; contacting the sensing probe with a sample to betested for the presence of the target molecule under a condition suchthat if the target molecule is present the sensing probe becomesassociated with the target molecule; exciting the sensing probe with anexcitation at about 800 nm; and detecting an emission at a shorterwavelength than 800 nm to determining the presence of the targetmolecule, wherein the upconversion luminescence nanoparticle comprises:a tri-doped core of β-NaYF₄: Ln_((i)), Ln_((j)), Ln_((k)); and anepitaxial shell of β-NaYF₄, wherein Ln_((i)) is Nd; Ln_((j)) is Yb; andLn_((k)) is Er or Tm.
 2. The method of claim 1, wherein the targetmolecule is a protein.
 3. The method of claim 2, wherein the targetmolecule is an antibody.
 4. The method of claim 2, wherein the targetmolecule is an enzyme.
 5. The method of claim 1, wherein the targetmolecule is a nucleic acid molecule.
 6. The method of claim 1, whereinLn_((k)) is Er.
 7. The method of claim 1, wherein Ln_((k)) is Tm.
 8. Themethod of claim 1, wherein the core has a dimension from about 1 nm toabout 100 nm; and the shell has a dimension from about 1 nm to about 50nm.
 9. The method of claim 1, wherein the upconversion luminescencenanoparticle further comprises a chemically modified surface, whereinthe surface has been modified by poly(acrylic acid).
 10. The method ofclaim 1, wherein the upconversion luminescence nanoparticle ischaracterized by an emission spectrum comprising a peak at about 540 nm,at about 525 nm or at about 474 nm.
 11. A method for detecting a targetmolecule in a sample, comprising: providing a sensing probe comprisingan upconversion luminescence nanoparticle having a constitutionalexcitation peak at about 800 nm, wherein the sensing probe is capable ofassociation with the target molecule; contacting the sensing probe witha sample to be tested for the presence of the target molecule under acondition such that if the target molecule is present the sensing probebecomes associated with the target molecule; exciting the sensing probewith an excitation at about 800 nm; and detecting an emission at ashorter wavelength than 800 nm to determining the presence of the targetmolecule, wherein the upconversion luminescence nanoparticle comprises:a core of β-NaYF₄: Ln_((j)), Ln_((k)); an epitaxial inner shell ofβ-NaYF₄: Ln_((i)), Ln_((j)), Ln_((k)); and an epitaxial outer shell ofβ-NaYF₄, wherein Ln_((i)) is Nd; Ln_((j)) is Yb; and Ln_((k)) is Er orTm.
 12. The method of claim 11, wherein Ln_((k)) is Er.
 13. The methodof claim 11, wherein Ln_((k)) is Tm.
 14. The method of claim 11, whereinthe core has a dimension from about 5 nm to about 100 nm; the innershell has a dimension from about 1 nm to about 20 nm; and the outershell has a dimension from about 1 nm to about 20 nm.
 15. The method ofclaim 11, wherein the upconversion luminescence nanoparticle furthercomprises a surface chemically modified by poly(acrylic acid).
 16. Themethod of claim 11, wherein the upconversion luminescence nanoparticleis characterized by an emission spectrum comprising a peak at about 540nm, at about 525 nm or at about 474 nm.
 17. A method forphoto-activating a protein in a sample, comprising: providing a sensingprobe comprising an upconversion luminescence nanoparticle having aconstitutional excitation peak at about 800 nm, wherein the sensingprobe is capable of association with a light-sensitive protein in thesample; contacting the sensing probe with the sample under a conditionsuch that if the light-sensitive protein is present the sensing probebecomes associated with the protein; exciting the sensing probe with anexcitation at about 800 nm; and activating the protein with an emissionat a shorter wavelength than 800 nm, wherein the upconversionluminescence nanoparticle comprises: a tri-doped core of β-NaYF₄:Ln_((i)), Ln_((j)), Ln_((k)); and an epitaxial shell of β-NaYF₄, whereinLn_((i)) is Nd; Ln_((j)) is Yb; and Ln_((k)) is Er or Tm.